Neuropeptides function peripherally as paracrine and endocrine factors to regulate diverse physiological processes and act as neurotransmitters and neuro-modulators. In a large majority of cases, the receptors which mediate signaling by neuropeptides are members of the superfamily of the G protein coupled seven membrane spanning receptors (Burbach and Meijer, Eur J Pharmacol, 227, 1–18, 1992). Neuropeptides have been documented to play important roles as autocrine/paracrine growth factors for human cancers (Rozengurt E, 1996, In Neuropeptides growth factors: Signaling pathways and role in cancer. In: Pusztai, L. L et al (editors)., Cell proliferation in cancer: Regulatory mechanisms of Neoplastic cell growth, Oxford, Oxford University press, page 247–259, 1996). The interruption of autocrine and paracrine neuropeptide signaling with specific antagonists or broad spectrum biased antagonists offer new therapeutic approaches to the treatment of cancer (Lynn E. H, Oncogene research, Vol 20, 1563–1569, 2001)
We have previously shown in our U.S. Pat. No. 6,156,725 and Australian Patent 707,158; (Mukherjee et al) that vasoactive intestinal peptide (VIP), somatostatin substance P and bombesin are secreted by some human tumor cells and that there are specific high affinity binding sites for these peptides on these cells. The four peptides were also shown to bind to tumor cells. The antagonist/analogs of these peptides were shown to have anti-proliferative activity on certain cancer cells, more specifically adenocarcinomas. A combination of the peptide antagonists/analogs was also shown to cause tumor regression in a nude mice xenograft model. It was hypothesized by us that there exists an autocrine mechanism for cell proliferation where the peptides are secreted by tumor cells and transduce cellular signals through specific cell surface receptors leading to cell proliferation. The analogs/antagonists to these peptides may then abrogate/block these cellular signals linked to proliferation. Further the antiangiogenic potential of the antagonist/analogs has also been described by us previously (Mukherjee et al; U.S. application Ser. No. 09/248,381 and PCT application WO 00/047221).
We have previously described in our U.S. patent (U.S. Pat. No. 6,316,414; Burman et al) novel peptides that are agonists to somatostatin and their use for the treatment of cancer. This invention particularly relates to the design and synthesis of novel analogs of somatostatin incorporating alpha, alpha-dialkylated amino acids in a site specific manner. The methods for the generation of these peptides, compositions containing the peptides and the pharmacological applications of these peptides especially in the treatment and prevention of cancer have also been described.
We have also described in our U.S. patent application Ser. Nos. (09/630,333, 09/630,345 and 09/629,642, Burman et al) novel antiproliferative analogs of VIP, bombesin and substance P that are useful in the treatment of cancer. These analogs incorporate α,α-dialkylated amino acids and show antiproliferative activity in a number of human tumor cell lines. Further, they caused partial tumor regression in nude mice xenografts when administered in a dose of approximately 1–25 microliter/mouse. Thus, when used individually, these peptides show moderate levels of cytotoxicity.
Neuropeptides and their analogs bind to specific high affinity transmembrane receptors on target cells to initiate a cascade of cytoplasmic signaling events. This includes the recruitment of several effector/adapter proteins initiating a cascade of protein-protein interactions. The binding of neuropeptides to their receptors causes the modulation of several cellular effector systems like adenylyl cyclase which cause alteration in the levels of second messenger molecules (Pimentel, In Growth factors and neoplasia, In Handbook of Growth factors, CRC Press, U.S.A, Vol 1, 329–337, 1994). The binding of peptide ligand to its receptor also causes the activation of intracellular protein kinases/phosphatases which are critical negative/positive regulators in the intracellular signaling pathways that result in growth factor specific cell responses like mitosis, differentiation, transformation or death. Several elements of this cascade have been directly/indirectly implicated in the malignant transformation and tumorogenesis of adenocarcinoma. The role of neuropeptides in cancer and cancer associated angiogenesis has been previously extensively reviewed. (Anticancer Res September–October 2000; 20(5A):3123–9); (Danesi R, Del Tacca M, Metabolism 1996 December; 45(8 Suppl 1): 49–50); (Woltering E A et al, J Surg Res 1991 March; 50(3): 245–251).
The adenocarcinomas express and secrete multiple growth factors viz. platelet derived growth factor (PDGF), epidermal growth factor (EGF) and transforming growth factor (TGF) alpha. The binding of growth factors to their respective receptors activates a cascade of intracellular pathways, specifically phosphorylation events mediated by protein kinases and phosphatases, which modulate the activity of a variety of cellular transcription factors. Aberrations in these signal-induced events are associated with cancer development and/or progression of cancer.
The cellular signaling mediated by receptors coupled to G proteins, as those for regulatory peptides are transduced through the cAMP-adenylyl cyclase system. The mitogenic response of the cells to growth factors and regulatory peptides is influenced by intracellular concentrations of cAMP, which in turn activates the cAMP dependent protein kinases (PKA), cAMP cooperates with a variety of hormones and growth factors to synergistically stimulate the proliferation of different type of eukaryotic cells.
The receptor tyrosine kinases (RTK) are transactivated by G protein coupled receptors (GPCR). Platelet derived growth factor (PDGF), epidermal growth factor (EGF) and insulin like growth factor 1 (IGF1) are tyrosine phosphorylated subsequent to GPCR activation. The phosphorylated growth factors in turn recruit multiple accessory proteins to activate the mitogen activated protein kinases (MAPK). Human adenocarcinomas have increased constitutive MAPK activity (Ostrowski et al, Br. J Cancer Vol 78, 1301–1306, 1997), and the blockade of this protein kinase suppresses tumour growth in vitro and in vivo (Sebolt-Leopold et al, Nature Medicine, Vol 5, 810–816, 1999). The MAP Kinase pathway is a crucial convergence point for many cytoplasmic signaling networks. MAP kinases form a family of Ser/Thr kinases, which can be activated by cellular growth factors. MAP Kinase lies downstream of the Ras-Raf oncogenic pathway and its activation leads to phosphorylation of nuclear transcription factors resulting in cell proliferation (Ostrowski et al., Br. J Cancer Vol 78, 1301–1306, 1997).
Membrane associated tyrosine phosphatases dephosphorylate specific targets, thus functionally opposing the action of tyrosine kinases. Inhibition of tyrosine phosphatase reversibly induces transformation of cultured cells in a dose dependent manner in vitro. Induction of tyrosine phosphatases in cancer cells leads to cell differentiation and reversal of transformed phenotype.
The p53 tumor suppressor protein plays a key role in the control of the normal cell cycle & apoptotic signalling. p53 gene mutations occur in approximately half of all malignancies from a wide variety of human tumors and are associated with poor prognosis and treatment (Kirch & Kastan, J. Clin Oncology, Vol 16, 3158–3168, 1998). Over expression of wild type p53 in cells induces cell cycle growth arrest, which in specific cases results in apoptosis.
Bcl-2 is the first member of a family of proteins shown to prevent death that is either p53 dependant or p53 independent (Peled et al, Cancer research, 56, 2148–2156, May 1, 1996). Overproduction of the bcl-2 protein also prevents cell death induced by nearly all cytotoxic anticancer agents frequently contributing to treatment failures in patients with several types of cancers (Reed et al, J. Cell Biochem, Vol 60, 23–32, 1996). The apoptotic fragmentation of the DNA is brought about by the sequential activation of the family of caspases.